TheranosticsFeb 16, 2026

Using nucleotide metabolism to influence base editing by glycosylase enzymes

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Changing nucleotide pools can steer glycosylase base editors toward more efficient B-to-A editing outcomes in mammalian cells.

Evidence

A genome-editing platform experiment showed that thymidine supplementation increased C-to-A, G-to-A, and T-to-A editing by up to 4-fold, 1.8-fold, and 1.8-fold, respectively, and improved A-product purity by up to 2.7-fold.

Caveat

The evidence is limited to cellular and disease-relevant SNV model editing outcomes, with no demonstration of clinical delivery, safety, or therapeutic efficacy.

Simplified

RATIONALE: Glycosylase-derived base editors enable transversion base substitutions, expanding the scope of genome engineering for both basic research and clinical applications. However, the variable outcomes and low efficiency of B (C/G/T)-to-A editing in mammalian cells hinder their broader utility, likely due to inefficient thymine translesion synthesis (TLS) across apurinic/apyrimidinic (AP) sites.

METHODS AND RESULTS: We developed a nucleotide metabolism-based strategy to enhance B-to-A editing by leveraging endogenous nucleotide metabolism. We showed that elevating intracellular deoxythymidine triphosphate (dTTP) levels via exogenous thymidine (dT) supplementation, which activates the thymidine kinase 1 (TK1)-dependent salvage pathway for the production of dTTP, increased C-to-A, G-to-A, and T-to-A editing efficiencies by up to 4-fold, 1.8-fold, and 1.8-fold, respectively, and improved A-product purity by up to 2.7-fold. Moreover, supplementation with dA increased T outcomes, albeit at a relatively modest level. In a disease-relevant single nucleotide variation (SNV) model, dT treatment enabled efficient generation of pathogenic mutations otherwise inaccessible to base editing.

CONCLUSION: Our findings establish metabolic modulation as a powerful means to control base editing outcomes and expand the functional capabilities of glycosylase-derived editors.